This project involves the isolation and characterization of Bacillus subtilis rpsD gene, encoding ribosomal protein S4, and the analysis of rpsD expression. A large proportion of the biosynthetic capacity of the bacterial cell is invested in the synthesis and maintenance of the machinery for protein synthesis. The mechanism for regulation of ribosome biosynthesis has been partially characterized in the Gram-negative bacterium Escherichia coli, and involves a complex negative feedback regulatory pattern to prevent accumulation of excess ribosomal components. It is not known whether similar systems are used in other organisms. B. subtilis is a Gram-positive spore-forming bacterium, evolutionarily distant from E. coli; of the Gram- positive bacteria, B. subtilis possesses the best-developed genetic system, and is well-suited for the analysis of ribosome biosynthesis because a large number of the genetic determinants for ribosomal components have been identified. However, little is known about the regulation of expression of ribosomal components. Mutations in the rpsD gene which alter the electrophoretic mobility of the S4 protein have been mapped, localizing the rpsD gene on the B. subtilis genetic map. Chromosomal insertions of transposon Tn917 located very close to the wild-type rpsD gene will be used to direct integration of specialized plasmid vectors into this region of the chromosome. Excision of the integrated plasmid along with flanking chromosomal DNA by digestion with various restriction endonucleases will permit cloning of chromosomal sequences in this region. The presence of the wild- type rpsD gene in cloned DNA will be determined by a transformation assay, using a recipient strain containing a mutant rpsD allele. The structure of the cloned DNA will be analyzed using restriction digestion analysis and hybridization experiments. The sequence of this region of the DNA will be determined, and potential regulatory regions and transcriptional units will be identified. S1 nuclease mapping experiments and gene fusions will be used to identify the start-points for transcription and translation, and to examine the regulation of S4 synthesis. These studies will localize regions important for regulation of expression; these sequences will be further analyzed by deletion and site-specific mutagenesis.